Human Muscle Development Research Articles

Engineered myovascular tissues for studies of endothelial/satellite cell interactions

In native skeletal muscle, capillaries reside in close proximity to muscle stem cells (satellite cells, SCs) and regulate SC numbers and quiescence through partially understood mechanisms that are difficult to study in vivo. This challenge could be addressed by the development of a 3-dimensional (3D) in vitro model of vascularized skeletal muscle harboring both a pool of quiescent SCs and a robust network of capillaries. Still, studying interactions between SCs and endothelial cells (ECs) within a tissue-engineered muscle environment has been hampered by the incompatibility of commercially available EC media with skeletal muscle differentiation. In this study, we first optimized co-culture media and cellular ratios to generate highly functional vascularized human skeletal muscle tissues (“myovascular bundles”) with contractile properties (∼10 mN/mm2) equaling those of avascular, muscle-only tissues (“myobundles”). Within one week of muscle differentiation, ECs in these tissues formed a dense network of capillaries that co-aligned with muscle fibers and underwent initial lumenization. Incorporating vasculature within myobundles increased the total SC number by 82%, with SC density and quiescent signature being increased proximal (≤20μm) to EC networks. In vivo, at two weeks post-implantation into dorsal window chambers in nude mice, vascularized myobundles exhibited improved calcium handling compared to avascular implants. In summary, we engineered highly functional myovascular tissues that enable studies of the roles of EC-SC crosstalk in human muscle development, physiology, and disease. Statement of significanceIn native skeletal muscle, intricate relationships between vascular cells and muscle stem cells (“satellite cells”) play critical roles in muscle growth and regeneration. Current methods for in vitro engineering of contractile skeletal muscle do not recreate capillary networks present in vivo. Our study for the first time generates in vitro robustly vascularized, highly functional engineered human skeletal muscle tissues. Within these tissues, satellite cells are more abundant and, similar to in vivo, they are more dense and less proliferative proximal to endothelial cells. Upon implantation in mice, vascularized engineered muscles show improved calcium handling compared to muscle-only implants. We expect that this versatile in vitro system will enable studies of muscle-vasculature crosstalk in human development and disease.

Human trapezius muscle development during the early fetal period.

This study aimed to observe human trapezius muscle (TpzM) development during early fetal period and apply diffusion tensor imaging (DTI) analysis to describe the muscle architecture that leads to physiological functions. Human embryonic and early fetal specimens were selected for this study. TpzM was first detected at Carnegie stage 20. The position of the TpzM changed with the formation of the scapula, clavicle, and vertebrae, which are its insertions and origins. DTI revealed the fiber orientation from each vertebral level to dissect each muscle. Fiber orientation in the ventral view gradually changed from the cervical to thoracic vertebrae, except for the middle part at which the insertions changed, which was almost similar in all early fetal specimens. The TpzM volume increased from C1 to C7 in the upper part, reached local maxima at C6 and C7 in the middle, and then decreased. These muscles can be categorized into three parts according to their insertions and presented with the features of each part. The fiber orientation and distribution of the three parts at the vertebral level were almost constant during the early fetal period. The border between the upper and middle parts was mainly located around the C6 and C7 vertebral levels, whereas the middle and lower parts were between the Th1 and Th2 vertebral levels. A three-dimensional change in the fiber orientation in the upper part of the TpzM according to the vertebral level was noticeable. Our data will help to elucidate the developmental processes of TpzM.

Single cell transcriptomic profiling identifies tumor-acquired and therapy-resistant cell states in pediatric rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric tumor that resembles undifferentiated muscle cells; yet the extent to which cell state heterogeneity is shared with human development has not been described. Using single-cell/nucleus RNA sequencing from patient tumors, patient-derived xenografts, primary in vitro cultures, and cell lines, we identify four dominant muscle-lineage cell states: progenitor, proliferative, differentiated, and ground cells. We stratify these RMS cells/nuclei along the continuum of human muscle development and show that they share expression patterns with fetal/embryonal myogenic precursors rather than postnatal satellite cells. Fusion-negative RMS (FN-RMS) have a discrete stem cell hierarchy that recapitulates fetal muscle development and contain therapy-resistant FN-RMS progenitors that share transcriptomic similarity with bipotent skeletal mesenchymal cells. Fusion-positive RMS have tumor-acquired cells states, including a neuronal cell state, that are not found in myogenic development. This work identifies previously underappreciated cell state heterogeneity including unique treatment-resistant and tumor-acquired cell states that differ across RMS subtypes.

An InDel in the Promoter of Ribosomal Protein S27-like Gene Regulates Skeletal Muscle Growth in Pigs

Genetic improvement of meat production traits has always been the primary goal of pig breeding. Geographical isolation, natural and artificial selection led to significant differences in the phenotypes of meat production traits between Chinese local pigs and Western commercial pigs. Comparative genomics and transcriptomics analysis provided powerful tools to identify genetic variants and genes associated with skeletal muscle growth. However, the number of available genetic variants and genes are still limited. In this study, a comprehensive comparison of transcriptomes showed that ribosomal protein S27-like (RPS27L) gene was highly expressed in skeletal muscle and up-regulated in Chinese local pigs when compared with Western commercial pigs. Functional analysis revealed that overexpression of RPS27L promoted myoblast proliferation and repressed differentiation in pig skeletal muscle cells. Conversely, the knockdown of RPS27L led to the inhibition of myoblast proliferation and the promotion of differentiation. Notably, a 13-bp insertion-deletion (InDel) mutation was identified within the RPS27L promoter, inserted in Chinese local breeds and predominantly deleted in Western commercial breeds. Luciferase reporter assay suggested this InDel modulated RPS27L expression by influencing transcription factor 3 (TCF3) and myogenic differentiation antigen (MYOD) binding to promoter. Furthermore, a positive correlation was observed between the expression of RPS27L expression and backfat thickness. Association studies demonstrated this InDel was significantly associated with the body weight of pigs at the age of 240 days. Together, our results suggested that RPS27L was a regulator of skeletal muscle development and growth, and was a candidate marker for improving meat production traits in pigs. This study not only provided a biomarker for animal breeding, but also was helpful for understanding skeletal muscle development and muscle-related disease in humans.

A human embryonic limb cell atlas resolved in space and time

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

Engineered skeletal muscle recapitulates human muscle development, regeneration and dystrophy.

Human pluripotent stem cell-derived muscle models show great potential for translational research. Here, we describe developmentally inspired methods for the derivation of skeletal muscle cells and their utility in skeletal muscle tissue engineering with the aim to model skeletal muscle regeneration and dystrophy in vitro. Key steps include the directed differentiation of human pluripotent stem cells to embryonic muscle progenitors followed by primary and secondary foetal myogenesis into three-dimensional muscle. To simulate Duchenne muscular dystrophy (DMD), a patient-specific induced pluripotent stem cell line was compared to a CRISPR/Cas9-edited isogenic control line. The established skeletal muscle differentiation protocol robustly and faithfully recapitulates critical steps of embryonic myogenesis in two-dimensional and three-dimensional cultures, resulting in functional human skeletal muscle organoids (SMOs) and engineered skeletal muscles (ESMs) with a regeneration-competent satellite-like cell pool. Tissue-engineered muscle exhibits organotypic maturation and function (up to 5.7±0.5mN tetanic twitch tension at 100Hz in ESM). Contractile performance could be further enhanced by timed thyroid hormone treatment, increasing the speed of contraction (time to peak contraction) as well as relaxation (time to 50% relaxation) of single twitches from 107±2 to 75±4ms (P<0.05) and from 146±6 to 100±6ms (P<0.05), respectively. Satellite-like cells could be documented as largely quiescent PAX7+ cells (75±6% Ki67- ) located adjacent to muscle fibres confined under a laminin-containing basal membrane. Activation of the engineered satellite-like cell niche was documented in a cardiotoxin injury model with marked recovery of contractility to 57±8% of the pre-injury force 21days post-injury (P<0.05 compared to Day 2 post-injury), which was completely blocked by preceding irradiation. Absence of dystrophin in DMD ESM caused a marked reduction of contractile force (-35±7%, P<0.05) and impaired expression of fast myosin isoforms resulting in prolonged contraction (175±14ms, P<0.05 vs. gene-edited control) and relaxation (238±22ms, P<0.05 vs. gene-edited control) times. Restoration of dystrophin levels by gene editing rescued the DMD phenotype in ESM. We introduce human muscle models with canonical properties of bona fide skeletal muscle in vivo to study muscle development, maturation, disease and repair.

Abstract IA014: Stem cell and developmental hierarchies in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and is comprised of at least two major molecular subtypes. Despite sharing features with skeletal muscle, the conservation of underlying cellular hierarchy with human muscle development and the identification of molecularly-defined tumor-propagating cells have not been reported. Using single-cell RNA sequencing of patient-derived RMS, DNA-barcode cell fate mapping, antibody enrichment and functional stem cell assays, and mouse xenograft modeling, we have uncovered tumor cell hierarchies in Fusion-negative (FN-) RMS that are shared with normal human muscle development. We also identified common developmental stages at which tumor cells become arrested. FN-RMS resemble early muscle found in embryonic and larval development, while fusion-positive (FP-) RMS express a highly specific developmental gene program found in muscle cells transiting from embryonic to fetal development at 7-7.75 weeks of age. FP-RMS also have neural-pathway enriched cell states, suggesting less-rigid adherence to muscle development hierarchies in this disease. Finally, we identify a new molecularly-defined tumor-propagating cell in FN-RMS that shares remarkable similarity to the newly described bi-potent, muscle mesenchyme stem/progenitor cell that makes both muscle and osteogenic cells. Citation Format: David M. Langenau, Yun Wei, Qian Qin, Luca Pinello. Stem cell and developmental hierarchies in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA014.

Copy number variation of EIF4A2 loci related to phenotypic traits in Chinese cattle.

BackgroundGenerally, copy number variation (CNV) is a large‐scale structural variation between 50 bp and 1 kb of the genome. It can affect gene expression and is an important reason for genetic diversity and phenotypic trait diversity. Studies have shown that the eukaryotic translation initiation factor 4A2 (EIF4A2) gene plays an essential role in muscle development in both humans and pigs. However, the influence of bovine EIF4A2’s copy number change on phenotypic traits has not been reported.ObjectivesTo detect the tissue expression profile of the EIF4A2 gene in adult cattle and individuals’ CNV type of variation. Then, we explored the correlation between EIF4A2‐CNV and growth traits in Chinese cattle breeds.MethodsReal‐time fluorescent quantitative reverse transcription PCR (qRT‐qPCR) was used to determine the expression profile of the EIF4A2 gene. Real‐time fluorescent quantitative PCR (qPCR) was used to detect the CNV type of bovine populations. Then, SPSS 26.0 was used for association analysis.ResultsIn this study, a total of 513 individuals in four cattle breeds (Qinchuan cattle [QC], Yunling cattle [YL], Pinan cattle [PN] and Jiaxian cattle [JX]) were detected for EIF4A2 gene's CNV. The results showed that EIF4A2‐CNV has an essential impact on hip width (HW) and rump length (RL) in QC, heart girth (HG), chest depth (CD) and RL in YL and HW in PN. However, it had no significant effect on JX.ConclusionsThe above results suggest that EIF4A2 gene's CNV can be used as a molecular marker for cattle breeding, which is helpful to accelerate the breeding of superior beef cattle breeds.

Single-cell analysis and functional characterization uncover the stem cell hierarchies and developmental origins of rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a common childhood cancer that shares features with developing skeletal muscle. Yet, the conservation of cellular hierarchy with human muscle development and the identification of molecularly defined tumor-propagating cells has not been reported. Using single-cell RNA-sequencing, DNA-barcode cell fate mapping and functional stem cell assays, we uncovered shared tumor cell hierarchies in RMS and human muscle development. We also identified common developmental stages at which tumor cells become arrested. Fusion-negative RMS cells resemble early myogenic cells found in embryonic and fetal development, while fusion-positive RMS cells express a highly specific gene program found in muscle cells transiting from embryonic to fetal development at 7-7.75 weeks of age. Fusion-positive RMS cells also have neural pathway-enriched states, suggesting less-rigid adherence to muscle-lineage hierarchies. Finally, we identified a molecularly defined tumor-propagating subpopulation in fusion-negative RMS that shares remarkable similarity to bi-potent, muscle mesenchyme progenitors that can make both muscle and osteogenic cells.

The road to physiological maturation of stem cell-derived cardiac muscle runs through the sarcomere

Recent advances the cardiac biomedical sciences have been propelled forward by the development and implementation of human iPSC-derived cardiac muscle. These notable successes notwithstanding, it is well recognized in the field that a major roadblock persists in the lack of full “adult cardiac muscle-like” maturation of hiPSC-CMs. This Perspective centers focus on maturation roadblocks in the essential physiological unit of muscle, the sarcomere. Stalled sarcomere maturation must be addressed and overcome before this elegant experimental platform can reach the mountaintop of making impactful contributions in disease pathogenesis, drug discovery, and in clinical regenerative medicine.

Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro.

Satellite cells (SC) are muscle stem cells that can regenerate adult muscles upon injury. Most SC originate from PAX7+ myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7+ myogenic precursors. We show that the PAX7+ cells generated in culture can produce myofibers and self-renew in vitro and in vivo Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.

4D formation of human embryonic forelimb musculature.

ABSTRACTThe size, shape and insertion sites of muscles enable them to carry out their precise functions in moving and supporting the skeleton. Although forelimb anatomy is well described, much less is known about the embryonic events that ensure individual muscles reach their mature form. A description of human forelimb muscle development is needed to understand the events that control normal muscle formation and to identify what events are disrupted in congenital abnormalities in which muscles fail to form normally. We provide a new, 4D anatomical characterisation of the developing human upper limb muscles between Carnegie stages 18 and 22 using optical projection tomography. We show that muscles develop in a progressive wave, from proximal to distal and from superficial to deep. We show that some muscle bundles undergo splitting events to form individual muscles, whereas others translocate to reach their correct position within the forelimb. Finally, we show that palmaris longus fails to form from early in development. Our study reveals the timings of, and suggests mechanisms for, crucial events that enable nascent muscle bundles to reach their mature form and position within the human forelimb.

Development of human limb muscles based on whole-mount immunostaining and the links between ontogeny and evolution.

We provide the first detailed ontogenetic analysis of human limb muscles using whole-mount immunostaining. We compare our observations with the few earlier studies that have focused on the development of these muscles, and with data available on limb evolution, variations and pathologies. Our study confirms the transient presence of several atavistic muscles - present in our ancestors but normally absent from the adult human - during normal embryonic human development, and reveals the existence of others not previously described in human embryos. These atavistic muscles are found both as rare variations in the adult population and as anomalies in human congenital malformations, reinforcing the idea that such variations/anomalies can be related to delayed or arrested development. We further show that there is a striking difference in the developmental order of muscle appearance in the upper versus lower limbs, reinforcing the idea that the similarity between various distal upper versus lower limb muscles of tetrapod adults may be derived.

Micropatterned substrates with physiological stiffness promote cell maturation and Pompe disease phenotype in human induced pluripotent stem cell-derived skeletal myocytes.

Recent advances in bioengineering have enabled cell culture systems that more closely mimic the native cellular environment. Here, we demonstrated that human induced pluripotent stem cell (iPSC)-derived myogenic progenitors formed highly-aligned myotubes and contracted when seeded on two-dimensional micropatterned platforms. The differentiated cells showed clear nuclear alignment and formed elongated myotubes dependent on the width of the micropatterned lanes. Topographical cues from micropatterning and physiological substrate stiffness improved the formation of well-aligned and multinucleated myotubes similar to myofibers. These aligned myotubes exhibited spontaneous contractions specifically along the long axis of the pattern. Notably, the micropatterned platforms developed bundle-like myotubes using patient-derived iPSCs with a background of Pompe disease (glycogen storage disease type II) and even enhanced the disease phenotype as shown through the specific pathology of abnormal lysosome accumulations. A highly-aligned formation of matured myotubes holds great potential in further understanding the process of human muscle development, as well as advancing in vitro pharmacological studies for skeletal muscle diseases.

Sialic acid catabolism by N-acetylneuraminate pyruvate lyase is essential for muscle function.

Sialic acids are important components of glycoproteins and glycolipids essential for cellular communication, infection, and metastasis. The importance of sialic acid biosynthesis in human physiology is well illustrated by the severe metabolic disorders in this pathway. However, the biological role of sialic acid catabolism in humans remains unclear. Here, we present evidence that sialic acid catabolism is important for heart and skeletal muscle function and development in humans and zebrafish. In two siblings, presenting with sialuria, exercise intolerance/muscle wasting, and cardiac symptoms in the brother, compound heterozygous mutations [chr1:182775324C>T (c.187C>T; p.Arg63Cys) and chr1:182772897A>G (c.133A>G; p.Asn45Asp)] were found in the N-acetylneuraminate pyruvate lyase gene (NPL). In vitro, NPL activity and sialic acid catabolism were affected, with a cell-type-specific reduction of N-acetyl mannosamine (ManNAc). A knockdown of NPL in zebrafish resulted in severe skeletal myopathy and cardiac edema, mimicking the human phenotype. The phenotype was rescued by expression of wild-type human NPL but not by the p.Arg63Cys or p.Asn45Asp mutants. Importantly, the myopathy phenotype in zebrafish embryos was rescued by treatment with the catabolic products of NPL: N-acetyl glucosamine (GlcNAc) and ManNAc; the latter also rescuing the cardiac phenotype. In conclusion, we provide the first report to our knowledge of a human defect in sialic acid catabolism, which implicates an important role of the sialic acid catabolic pathway in mammalian muscle physiology, and suggests opportunities for monosaccharide replacement therapy in human patients.

Biallelic mutation of UNC50, encoding a protein involved in AChR trafficking, is responsible for arthrogryposis.

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Homozygosity mapping of disease loci combined with whole exome sequencing in a consanguineous family presenting with lethal AMC allowed the identification of a homozygous frameshift deletion in UNC50 gene (c.750_751del:p.Cys251Phefs*4) in the index case. To assess the effect of the mutation, an equivalent mutation in the Caenorhabditis elegans orthologous gene was created using CRISPR/Cas9. We demonstrated that unc-50(kr331) modification caused the loss of acetylcholine receptor (AChR) expression in C. elegans muscle. unc-50(kr331) animals were as resistant to the cholinergic agonist levamisole as unc-50 null mutants suggesting that AChRs were no longer expressed in this animal model. This was confirmed by using a knock-in strain in which a red fluorescent protein was inserted into the AChR locus: no signal was detected in unc-50(kr331) background, suggesting that UNC-50, a protein known to be involved in AChR trafficking, was no longer functional. These data indicate that biallelic mutation in the UNC50 gene underlies AMC through a probable loss of AChR expression at the neuromuscular junction which is essential for the cholinergic transmission during human muscle development.

A defect in myoblast fusion underlies Carey-Fineman-Ziter syndrome

Multinucleate cellular syncytial formation is a hallmark of skeletal muscle differentiation. Myomaker, encoded by Mymk (Tmem8c), is a well-conserved plasma membrane protein required for myoblast fusion to form multinucleated myotubes in mouse, chick, and zebrafish. Here, we report that autosomal recessive mutations in MYMK (OMIM 615345) cause Carey-Fineman-Ziter syndrome in humans (CFZS; OMIM 254940) by reducing but not eliminating MYMK function. We characterize MYMK-CFZS as a congenital myopathy with marked facial weakness and additional clinical and pathologic features that distinguish it from other congenital neuromuscular syndromes. We show that a heterologous cell fusion assay in vitro and allelic complementation experiments in mymk knockdown and mymkinsT/insT zebrafish in vivo can differentiate between MYMK wild type, hypomorphic and null alleles. Collectively, these data establish that MYMK activity is necessary for normal muscle development and maintenance in humans, and expand the spectrum of congenital myopathies to include cell-cell fusion deficits.

Connecting Sarcomere Protein Mutations to Pathogenesis in Cardiomyopathies: The Development of "Disease in a Dish" Models.

Recent technological and protocol developments have greatly increased the ability to utilize stem cells transformed into cardiomyocytes as models to study human heart muscle development and how this is affected by disease associated mutations in a variety of sarcomere proteins. In this perspective we provide an overview of these emerging technologies and how they are being used to create better models of “disease in a dish” for both research and screening assays. We also consider the value of these assays as models to explore the seminal processes in initiation of the disease development and the possibility of early interventions.

Sodium nitrate decreases agrin-induced acetylcholine receptor clustering.

BackgroundHumans are exposed to nitrate predominantly through diet with peak plasma concentrations within an hour after ingestion, but additional exposure is obtained from the environment, and minimally through de novo synthesis. Higher nitrate consumption has been associated with methemoglobinemia, spontaneous abortions, atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, amyotrophic lateral sclerosis, and neural tube defects. However, skeletal muscle development has not been examined.MethodsC2C12 skeletal muscle cell cultures were maintained, myoblasts were fused into myotubes, and then cultures were exposed to motor neuron derived agrin to enhance acetylcholine receptor (AChR) clustering. Untreated cultures were compared with cultures exposed to sodium nitrate at concentrations ranging from 10 ng/mL–100 μg/mL.ResultsThe results reported here demonstrate that 1 μg/mL sodium nitrate was sufficient to decrease the frequency of agrin-induced AChR clustering without affecting myotube formation. In addition, concentrations of sodium nitrate of 1 μg/mL or 100 μg/mL decreased gene expression of the myogenic transcription factor myogenin and AChR in correlation with the agrin-induced AChR clustering data.ConclusionsThese results reveal that sodium nitrate decreases the frequency of agrin-induced AChR clustering by a mechanism that includes myogenin and AChR gene expression. As a consequence sodium nitrate may pose a risk for skeletal muscle development and subsequent neuromuscular synapse formation in humans.

Molecular microevolution and epigenetic patterns of the long non-coding gene H19 show its potential function in pig domestication and breed divergence.

BackgroundThe domestic pig Sus scrofa domesticus originated from the wild boar S. scrofa about 10,000 years ago. During domestication, drastic morphological, physiological, and behavioral changes developed between domestic pigs and wild boars through artificial and natural selection. The long non-coding RNA (lncRNA) H19, which is located within the imprinting gene cluster H19-IGF2, plays an important role in regulating muscle development in humans and mice. This study systematically analyzed the molecular evolution of H19 and its possible epigenetic changes during pig domestication and breeding to explore the genetic and epigenetic contributions of H19 to pig domestication.ResultsThe molecular evolution of H19 was initially analyzed on a large phylogenetic scale. Results showed that the gene was highly conserved within a broad range, especially in the 5′ terminal sequence. The molecular evolution of the gene was then analyzed using published re-sequencing data of 30 wild boars from Tibet, 3 wild boars from Sichuan, and 15 native pigs from other regions in China. Eight polymorphic sites were identified, and the nucleotide diversity (π) value within the H19 gene body was significantly higher (Z-test, P < 0.05) in domesticated pigs than in wild pigs. However, no significant divergence occurred between domesticated and wild pigs. Single nucleotide polymorphisms in the 3′ terminal sequence were surveyed in other Chinese local breeds and foreign pig breeds. We observed a consistently higher diversity in domesticated pigs than in wild pigs. The methylation pattern of the H19 gene in pigs was subsequently analyzed using published methylated DNA immunoprecipitation data and an unpublished single-base resolution liver methylome. Analysis results showed distinct methylation levels in some tissues. Among the samples surveyed, Landrace showed the lowest methylation level, followed by the Guizhou wild boar, whereas the Enshi pig exhibited the highest methylation level in the 2 kb upstream region of the H19 gene. Liver transcriptome data suggested that Landrace harbored the highest expression of the H19 gene, followed by the Guizhou wild boar, whereas the Enshi pig harbored the lowest expression of the gene. Differential methylation sites (DMSs) among the three breeds were mainly identified in the 2 kb upstream region of the H19 gene. In the Enshi pig, we detected allele-specific methylation (ASM) regions in the 2 kb upstream region of the H19 gene. Most of the DMSs in the upstream 2 kb region of the gene were also located in the ASM region in this breed.ConclusionsMolecular analyses suggest that the H19 gene was highly conserved during large-scale evolution and exhibited genotype differentiation during domestication and breed differentiation. The drastic diversity pattern between domestic and wild pigs in the H19 gene body, which was highly conserved during large-scale evolution, suggests that this gene might have played roles in the breed differentiation of domestic pigs. Methylation analysis indicates an opposite epigenetic regulation direction between Chinese and European pig (EU) domestication, which resulted in opposite expression changes in this gene between the two domesticated groups. Our preliminary analyses on DMSs among different pig breeds and ASM imply that imprinting was associated with methylation differences. This study systematically demonstrates the genetic and epigenetic patterns of H19 during pig domestication and provide valuable cues and basis for further research on the function of H19 in pig domestication.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0657-5) contains supplementary material, which is available to authorized users.